Rath Subhashree, Paul Manish, Behera Hemanta Kumar, Thatoi Hrudayanath
Department of Biotechnology, Maharaja Sriram Chandra Bhanja Deo University, Takatpur, Baripada, Odisha, 757003, India.
J Genet Eng Biotechnol. 2022 Jan 3;20(1):2. doi: 10.1186/s43141-021-00284-2.
Lignin is a complex polymer of phenyl propanoid units found in the vascular tissues of the plants as one of lignocellulose materials. Many bacteria secrete enzymes to lyse lignin, which can be essential to ease the production of bioethanol. Current research focused on the study of ligninolytic bacteria capable of producing lignin peroxidase (LiP) which can help in lignin biodegradation and bioethanol production. Ligninolytic bacterial strains were isolated and screened from the soil samples of Simlipal Biosphere Reserve (SBR), Odisha (India), for the determination of their LiP activity. Enzymatic assay and optimization for the LiP activity were performed with the most potent bacterial strain. The strain was identified by morphological, biochemical, and molecular methods.
In this study, a total of 16 bacteria (Simlipal ligninolytic bacteria [SLB] 1-16) were isolated from forest soils of SBR using minimal salt medium containing lignin. Out of the 16 isolates, 9 isolates showed decolourization of methylene blue dye on LB agar plates. The bacterial isolates such as SLB8, SLB9, and SLB10 were able to decolourize lignin with 15.51%, 16.80%, and 33.02%, respectively. Further enzyme assay was performed using HO as substrate and methylene blue as an indicator for these three bacterial strains in lignin containing minimal salt medium where the isolate SLB10 showed the highest LiP activity (31.711 U/mg). The most potent strain, SLB10, was optimized for enhanced LiP enzyme activity using response surface methodology. In the optimized condition of pH 10.5, temperature 30 °C, HO concentration 0.115 mM, and time 42 h, SLB10 showed a maximum LiP activity of 55.947 U/mg with an increase of 1.76 times from un-optimized condition. Further chemical optimization was performed, and maximum LiP activity as well as significant dye-decolourization efficiency of SLB10 has been found in bacterial growth medium supplemented individually with cellulose, yeast extract, and MnSO. Most notably, yeast extract and MnSO-supplemented bacterial culture medium were shown to have even higher percentage of dye decolourization compared to normal basal medium. The bacterial strain SLB10 was identified as Bacillus mycoides according to morphological, biochemical, and molecular (16S rRNA sequencing) characterization and phylogenetic tree analysis.
Result from the present study revealed the potential of Bacillus mycoides bacterium isolated from the forest soil of SBR in producing LiP enzyme that can be evaluated further for application in lignin biodegradation and bioethanol production. Scaling up of LiP production from this potent bacterial strain could be useful in different industrial applications.
木质素是一种由苯丙烷单元组成的复杂聚合物，作为木质纤维素材料之一存在于植物的维管组织中。许多细菌会分泌酶来分解木质素，这对于简化生物乙醇的生产至关重要。目前的研究集中在能够产生木质素过氧化物酶（LiP）的木质素分解细菌上，这种酶有助于木质素的生物降解和生物乙醇的生产。从印度奥里萨邦西姆利帕尔生物圈保护区（SBR）的土壤样本中分离并筛选出木质素分解细菌菌株，以测定它们的LiP活性。对最具活性的细菌菌株进行了LiP活性的酶促测定和优化。通过形态学、生物化学和分子方法对该菌株进行了鉴定。
在本研究中，使用含有木质素的基本盐培养基从SBR的森林土壤中总共分离出16株细菌（西姆利帕尔木质素分解细菌[SLB]1 - 16）。在这16株分离菌中，有9株在LB琼脂平板上表现出亚甲基蓝染料的脱色。SLB8、SLB9和SLB10等细菌分离株分别能够使木质素脱色15.51%、16.80%和33.02%。在含有木质素的基本盐培养基中，以H2O2为底物、亚甲基蓝为指示剂对这三株细菌菌株进行了进一步的酶活性测定，其中分离株SLB10表现出最高的LiP活性（31.711 U/mg）。使用响应面法对最具活性的菌株SLB10进行了优化以提高LiP酶活性。在pH 10.5、温度30°C、H2O2浓度0.115 mM和时间42小时的优化条件下，SLB10表现出最大LiP活性55.947 U/mg，比未优化条件下增加了1.76倍。进行了进一步的化学优化，发现在分别添加纤维素、酵母提取物和MnSO4的细菌生长培养基中，SLB10具有最高的LiP活性以及显著的染料脱色效率。最值得注意的是，与正常基础培养基相比，添加酵母提取物和MnSO4的细菌培养基显示出更高的染料脱色百分比。根据形态学、生物化学和分子（16S rRNA测序）特征以及系统发育树分析，细菌菌株SLB10被鉴定为蕈状芽孢杆菌。
本研究结果揭示了从SBR森林土壤中分离出的蕈状芽孢杆菌在产生LiP酶方面的潜力，该酶可进一步评估其在木质素生物降解和生物乙醇生产中的应用。扩大这种强效细菌菌株的LiP生产规模可能在不同的工业应用中有用。
